The selectivity of anti-cancer drugs is poor and most of the drugs used for treatment have dose limiting toxicity (typically bone marrow toxicity but other tissues are affected depending on the anti-cancer drug). The use of doxorubicin, for example, is dose limited due to its cardiac toxicity and its myelosuppressive effect. Numerous attempts have been made to isolate related anthracycline drugs which show improved properties but doxorubicin and daunorubicin remain two of the most useful drugs for treatment of cancers and leukemias (Young, R C et al, New England J Medicine 1981, 305, 139-153; Zunino F and Capranico G, Anti-Cancer Drugs Design 1990, 5, 307-317; EP 0 441 218 A2).
The most interesting drugs for use as prodrugs are those which are toxic at low levels with IC50 (inhibitory concentration causing 50% inhibition of growth)&lt;10.sup.-5 M. Examples of such agents include morpholinyl anthracyclines (Streeter D G et al, Cancer Chemother Pharmacol 1985, 14, 160-164; U.S. Pat. No. 4,301,277), barminomycins (Uchida T, et al, The J of Antibiotics, 1988, XLI, 404-408) actinomycin D, and anthracycline analogs bearing latent alkylating substituents (U.S. Pat. No. 5,196,522). Morpholinyl anthracyclines such as compound 1 (see below) can dissociate in solution to form the reactive iminium compound 2; and compound 3, an anthracycline analog bearing a latent aldehyde, can undergo hydrolysis of the diacetoxy group by esterases in vivo followed by cyclization to form the analogous iminium compound 4. ##STR1##
Reactive iminium ions are formed by the reaction of amines with carbonyl groups such as aldehydes and ketones.
The need for more toxic agents as prodrugs has been suggested by a number of workers in this area (Henle K J, et al Radiation Res, 1988, 115, 373-386). Attempts have also been made to improve the properties of the anthracycline cancer drugs by making glycoside modifications to generate activatable prodrugs (EP 0 441 218 A2, Leenders Ruben G G et al Tet Lett. 1995, 36, 1701-1704, EP 0 565 133 A2, WO 92/19639). These anthracycline glycosides do not make use of the more potent anthracyclines resulting in less desirable anthracycline prodrugs due to the clinical problems of large doses and increased cost of synthesis.
Antibodies which are specific for tumors are well known in the art. Tumor specific antibodies have been used to target toxins in attempts to develop cancer therapies.
The production of drug antibody conjugates has been achieved with some success in vitro but with disappointing results in tumor bearing mice and clinical studies (Garnett M C, et al Int J Cancer 1983, 31 661-670, Embleton M J et al Br J Cancer 1983, 47 43-49).
Attempts have been made to improve selective delivery of cytotoxic agents to tumors using antibodies coupled to enzymes. Conjugation of enzymes such as ricin and other ribosome inactivating proteins to antibodies has been used to target enzymes to tumors. In these studies, the enzyme is also the active toxin entering the cell and catalytically inactivating it by modification of the ribosomes (Moller G, Immunol Rev 1982;62).
In other studies, attempts have been made to use the activities of enzymes conjugated to targeting- antibodies to generate cytotoxic agents for targeted tumor killing. The early work on this principle used glucose oxidase as the enzyme (Philpott G D, et al J Immunology 1973, 111, 921-929). See also Parker et al, 1975 Proc Nat Acad Sci USA 72, 338-342.
WO 87/03205 discloses enzyme-coupled antibody, in which the enzyme is characterized by its ability to catalyze reactions which result in the death of cells bearing antigenic sites which the antibody can bind. See also U.S. Pat. No. 4,975,278. In animal studies, tumor regressions were seen with the targeting of the CC 49 anti tag 72 antibody as a conjugate to beta-lactamase to tumors followed by the treatment of the animals with a vinblastine prodrug substrate for beta-lactamase, Meyer et al (Cancer Res 1993 53, 3956-3963). In these studies, in addition to the antibody-enzyme conjugates, the drug antibody conjugates were evaluated and shown to be relatively ineffective and required very large doses of antibody when compared to enzyme activation.
Targeting of glycosidic enzymes has also been successfully used. In one example, a tumor specific antibody was chemically cross linked to the enzyme E. coli beta-glucuronidase and used to target a rat hepatoma cell line. This targeted beta-glucuronidase was used to activate p-di-2-chloroethylaminophenyl-beta-D-glucuronide prodrug to its active drug N,N-di-(2-chloroethyl) 4-hydroxyaniline. (Wang S M et al Cancer Res 1992 Aug. 15;52(16):4484-91).
In a further demonstration of this approach, a pan carcinoma antibody was chemically linked to E. coli beta-glucuronidase generating a conjugate which was able to specifically target various carcinoma cells. The prodrug used in this study was a glucuronide of epirubicin which resulted in a detoxification of the parent drug up to 1000 fold. This glucuronide was isolated from the urine of patients treated with epirubicin. The in vitro data demonstrated good levels of activation and cytotoxicity using these conjugates (Haisma H J et al Br J Cancer 1992 September;66(3):474-8).
In another study making use of (i) a conjugate of E. coli beta (galactosidase to an anti-CEA antibody Col1 and (ii) a galactoside of 5-fluorouridine, targeted activation was also demonstrated (Abraham R et al 1994 Cell Biophysics 24/25, 127-133).
Prodrug approaches have also been used clinically making use of the prokaryotic enzyme carboxypeptidase-G2 fused to anti CEA antibodies (Bagshawe K D et al Br J Cancer 1988 58:700-703). In a study aimed at producing a less immunogenic antibody fusion, which may have advantages over mouse antibodies and bacterial enzymes, a fusion with the human beta glucuronidase was made to a humanized anti CEA antibody. This was achieved by making a genetic construction allowing reproducible production of the therapeutic antibody (Bosslet K et al Br J Cancer 1992 65:234-238, EP 0501215 A2). This humanized anti CEA antibody human beta glucuronidase fusion protein has been demonstrated to activate a glucuronide of doxorubicin (see WO 92/19639) in tumor bearing mice to achieve some reduction in tumor growth and 10 fold higher levels of doxorubicin in the tumor (Bosslet K et al, 1994 Cancer Res. 54, 2151-2159). In an approach similar to that described by Bosslet et al., the use of human antibodies and human lysozyme has also been proposed to reduce the potential problems associated with immunogenic antibodies and enzymes (WO 90/07929).
The potential for the use of exogenous glycosidic enzymes in a non targeted format has been investigated (Tshiersch B, Schwabe K, Sydow G, and Graffi A Cancer Treat Rep 1977 61:1489-1493). In this study a combination of alpha-L-arabinofuranosidase from Aspergillus niger was used in combination with a prodrug form of beta-peltatin A, beta-peltatin A-alpha-L-arabinofuranoside. The aim of this approach was to make use of the lower pH optimum for the alpha-L-arabinofuranosidase to develop selective activation in tumors based on the lower pH found in tumors. This group also made use of the ability to affect the tumor pH by glucose infusion.
The potential for the use of endogenous glycosidic enzymes in a non targeted format has been investigated. U.S. Pat. Nos. 4,327,074; 4,337,760; 4,481,195; 4,584,368; and 5,005,588 describe the potential of using beta-glucuronidase activity present in tumors. The inventors note that the effect can be enhanced by the use of glucose and alkalinization to increase the differences in pH between the tumor and the normal tissues. The use of glucose allows the tumor pH to be lowered significantly and the use of a base such as sodium bicarbonate allows the urine pH and other areas of normal tissue to remain at pH in the range of 7.4. The lowering of the tumor pH can be as much as 0.5 pH unit in some cases (Cancer Res 49, 4373-4384, 1989). U.S. Pat. No. 4,248,999 discloses the use of 5-fluorouracil as a glucuronide (and other glycosides) by linking it to the C6 position of the uracil ring. Protocols for the improvement of therapy with glucuronide prodrugs have also been suggested which make use of the potential for endogenous glucuronidase activity, increasing the whole body pH and lowering the tumor pH.
The potential of using virus and/or nucleic acid targeted prodrug activation has also been investigated. In an example of this approach, the enzyme cytosine deaminase has been targeted using a retroviral vector to achieve the selective delivery and activation of the prodrug 5-fluorocytosine. This has been demonstrated with the generation of retroviral vectors which have incorporated the cytosine deaminase gene from yeast under control of the CEA promoter (Huber B E et al, 1993, Cancer Res 53, 4619-4626). In a similar approach the herpes simplex virus thymidine kinase (HSV-tk) has been incorporated into retroviral vectors to activate ganciclovir to its toxic phosphorylated form (J. Michael DiMaio et al 1994, Surgery 116, 205-213). Other viruses may be used in this targeting approach such as adenovirus, fowlpox, newcastles disease. These viruses are being explored in the treatment of cancer. The delivery of the virus may be direct through the use of an infectious particle which optionally has been engineered to have a selective tissue tropism (i.e., by inclusion of antibody binding domains (Chu et al, J Virol 1995, 69 2659-63)). In an alternative method the virus is targeted by the use of other vehicles such as liposomes in either a targeted (by binding moieties, i.e., antibodies) or untargeted fashion (Bichko V et al 1994, J Virology 68, 5247-5252). The targeting and delivery of genes to activate prodrugs can also occur via the delivery of DNA (not in the form of a virus). An encapsulation method can be used for delivery either via liposomes or through the use of viral like particles to package the DNA as has been demonstrated with a number of E. coli viruses.